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HKL2MAP - Solving structure with HKL2MAP (SHELX) program suit
- Protein crystallography

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Special:

   - Programs for crystallography
       - ADSC-software
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       - HKL2000 package
       - HKL2MAP
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Xtal community:

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About HKL2MAP

HKL2MAP is very convenient shell to run all shelx programs, it will allow so save hours of file editing. HKL2MAP was developed by Thomas Pape and Thomas R. Schneider in 2003. The latest available version 0.2 is dated by 2005. For new version of this program please contact directly to the developers http://schneider.group.ifom-ieo-campus.it/hkl2map/ If you've use this program in your work please refers to:
Thomas Pape & Thomas R. Schneider HKL2MAP: a graphical user interface for phasing with SHELX programs J. Appl. Cryst. 37:843-844 (2004).

Also please refer to shelx references as follows:
For SHELXC: Sheldrick GM (2003). Gottingen University.
For SHELXD: Schneider TR, Sheldrick GM (2002). Substructure solution with SHELXD, Acta Cryst. D58:1772-1779.
For SHELXE: Sheldrick GM (2002). Macromolecular phasing with SHELXE, Z. Kristallogr. 217:644-650.

HKL2MAP is designed for solving structure with SAD/MAD as well as with SIR/SIRAS data by preparing command files and running SHELX program.

Step by step guide how to run HKL2MAP

In this chapter we will describe solving of the high resolution structure with HKL2MAP with using of MAD data, collected from proteins crystals with Se-Met (Selenomethionine substitution).

Data collection statistics:
ParametersNative proteinSeMet protein
Resolution (Å)20-1.020-2.5
λ Remote (Å)
λ Peak (Å)
λ Inflation (Å)

Protein: 210 residues with 6 methionine residues substituted by selenomethionine residues. All data sets were processed with HKL2000 data processing software with all diffraction data stored in SCA format.
Step Operations Screenshot Explanation
1 Start HKL2MAP
Type in command shell:
hkl2map<ENTER> 		hkl2map start Fig: 01 HKL2MAP initial window.
2 Project name
Type:
Project Name <ENTER>
We do recommend using unique name for each program run. We will name our project example 		hkl2map project name Fig: 02 HKL2MAP with Project Name example
3 Type of experiment - MAD
Select type of your experiment (MAD in our example) 		hkl2map MAD Fig: 03 HKL2MAP window for MAD experiment
4 Experimental data
Fill in your experimental data as follows:
Native in: usually high resolution native data set.
Peak in: Anomalous diffraction Peak wavelength.
Infl. in: Anomalous diffraction Inflation wavelength.
HRem in: Anomalous diffraction high energy remote wavelength.
LRem in: Anomalous diffraction low energy remote wavelength.
 hkl2map shelxc ready to run Fig: 04 HKL2MAP window everything ready for runing SHELXC
If you do not have some of these data, not a problem, just enter what you've got. Also please check the cell size and space group. With space group you also need to tick box "confirmed". Press "run SHELXC" hkl2map successful SHELXC output Fig: 04a HKL2MAP successful SHELXC output
5 Heavy atom search with SHELXD program
Close SHELXC window and open SHELXD window by clicking special buttons. In this window you need to specify which atoms you've got in your asymmetric cell, resolution of your anomalous data and most important question - number of tries. For number of atoms it is better to use slightly higher value, it will help to estimate the quality of solution. In most cases number of cycles should be around 20-30. After setting of all parameters, press "run SHELXD". 		hkl2map Shelxd Fig: 05 HKL2MAP, ShelxD window for MAD experiment
6 SHELXD log file analysis
Look at the image by pressing "view graphics" -> menu display -> Site occupancy vs. Peak Number
Occupancy of heavy atom sites can unambiguously tell you the number of Selenium atoms in the asymmetric unit. 		Site occupancy vs. Peak Number Fig: 06 Occupancy of heavy atom sites
7 ShelxE - phasing and density modification
Now we presume that in previous run SHELXD produce some solutions. Switch to SHELXE window.
In this window it is necessary to shows how many cycles of density refinement it is necessary to carry out. Usually 20 will be enough. Most important part - fractional solvent content. Uses button "estimate the solvent content" in order to receive more accurate value - this is very crucial for further calculations.
If you've use native data - you need to specify is there any heavy atoms in your native structure.
Another important parameter - enantiomorphs. SHELXD can not determine the hand of solution. This can be only determined at this stage. So for the first run use "try both enantiomorphs" and then select proper one. After all these settings do press "run SHELXE". 		hkl2map Shelxe Fig: 07 HKL2MAP, ShelxE window
8 Final analysis of SHELXE output
During SHELXE calculations it is possible to do "view graphics" and usually the enantiomorph selection will be very clear. In our case the original enantiomorph is correct and we can remove inverted structure. It is possible to stop calculation by pressing "finish inverted". The good sign of finishing of density modification is the approximation of the graph Contacts vs. Cycle to the constant value. If during calculations your graph is not become constant at the final cycles, restart SHELXE with bigger number of cycles.
At the end you will have two main files which will be necessary for further job:
example.phs - file with phases calculated from this experiment (or example_1.phs for inverted hand)
example_fa.pdp - PDB file with coordinated of all heavy atoms. It is recommended to manually shrink all weak atoms and leave only strong one with high occupancy. From previous analysis we know (see pp. 6) that in our case we've got only 5 atoms. 		Contrast vs. Cycle for density modifications Fig: 08 Contrast vs. Cycle for density modifications in ShelxE
9 Visual presentation
Experimental phases form example.phs can be directly converted to the electron density map. Easiest way to do this - load this file to graphical program coot. Detailed description of this program will be given in other chapter, but here we will give you a crucial point. This is necessary to perform proper FFT map calculation based on your phases. Please add cryst1 card to your PDB file as follows:
CRYST1 37.000 45.400 111.400 90.00 90.00 90.00 P 21 21 21

Load your PDB (example_fa.pdb) and experimental data set (example.phs) into coot and enjoy your map. If you've got good phasing power from your Selenium atoms, your map will be sufficient for model building. 		SeMet electron density
Phenylalanine electron density
beta-strands electron density 		Fig: 09 MAD electron density maps calculated from SeMet phases with 1.5σ cut-off, 1.1 Å resolution.
a. SeMet residue with positions of experimentally identified Selenium atom;
b. Phenylalanine moiety;
c. Group of beta-strands, tryptophan and tyrosine moieties.
10 File conversion
For many calculations it is necessary convert file with experimental phases into MTZ (internal CCP4 data format) format. It is very easy to do with f2mtz program with following command file:
          
          f2mtz   HKLIN  example.phs    HKLOUT example.mtz << finish
          TITLE  example data set
          CELL   37.100   45.400  111.400  90.00  90.00  90.00
          SYMM   19
          LABOUT H K L FP FOMS PHIS SIGFP
          CTYP   H H H F  W    P     Q
          END
          finish


Following script will allow you to calculate electron density map for asymmetric unit in CCP4 format:

          fft HKLIN example.mtz MAPOUT example.map << finish
          title example map calculation
          xyzlim asu
          scale F1 1.0
          labin F1=FP SIG1=SIGFP PHI=PHIS W=FOMS
          end
          finish


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